The invention relates to novel nanoscale particles, methods of making same and uses thereof. More particularly, the invention relates to nanoscale particles which optionally are encased in a shell comprising one or more virion coat proteins, and methods associated therewith. In particular, the invention relates to virion-constrained nanoparticles comprising an inorganic, organic and/or organo-metallic material surrounded by a shell of one or more virion coat proteins. The invention further relates to methods of producing nanoscale particles, optionally through the use of controlled gating.
Ultrafine particles are useful in the production of many materials ranging, for example, from coatings, particularly coatings of one or more layers, to high performance lubricants, and from electronic devices to therapeutic delivery systems. Traditionally, fine particles have been prepared by grinding larger particles. However, such grinding results in a heterogeneous mix of particle sizes and shapes, and thus limits the usefulness of such particles. Such mixes can be further fractionated, for example, by passage though one or more sieves. In this case, the fractions collected may be in a certain size range, but within that range the size and shape distribution remains heterogeneous. Moreover, this additional size selection may result in a large amount of material that is discarded. Due to the disparity in particle shapes and sizes, discontinuities, stresses, frictions, etc. may arise in the resultant material, layer, lubricant, etc. for which the particles are employed. Thus, even after the expenditure of much effort in the prior art, suitable particles for high performance and high tolerance applications could not heretofore be reliably and economically produced by grinding methods.
Attempts to circumvent these problems have met with limited success in the past. These alternative approaches have included condensation of vaporized atoms and controlled precipitation of solutes out of solutions. In the case of precipitation where seed particles are used, the heterogeneity of the seed particles themselves render mixtures that are polydisperse. There is thus a need in the art for monodisperse particles of a desired size and/or shape.
Bunker, et al ., "Ceramic Thin-Film Formation on Functionalized Interfaces Through Biomimetic Processing" Science 264: 48-55 (1994), discloses high density polycrystalline films of oxides, hydroxides and sulfides. These films are disclosed to be useful in a wide variety of applications. The films are prepared using substrates having functionalized surfaces. These surfaces are given a ceramic coating by the process of nucleation and particle growth mechanisms.
Aksay, et al., "Biomimetic Pathways for Assembling Inorganic Thin Films," Science 273: 892 898 (1996) discloses a process whereby a supramolecular assembly of surfactant molecules at an organic-inorganic interface to template for condensation of an inorganic silica lattice. The technique is thought to be useful in the synthesis of inorganic composites with designed architecture at the nanometer scale.
Huo, et al., "Generalized Synthesis of Periodic Surfactant/Inorganic Composite Materials" Nature 368: 317-321 (1994), discloses the direct co-condensation of anionic inorganic species with cationic surfactants and the cooperative condensation of cationic inorganic species with anionic surfactants. The cooperative assembly of cationic inorganic species with cationic surfactants is also disclosed. The main driving force for this self-assembly is thought to be electrostatic. The technique is useful for synthesis of several different mesostructured phases.
Evans et al., "Biomembrane Templates for Nanoscale Conduits and Networks," Science 273: 933-935 (1996) discloses the production of solid phase networks and conduits through the use of photochemical polymerization of long (20 to 200 nm) nanotubes. Nanotubes are formed by the mechanical retraction of a "feeder" vesicle after molecular bonding to a rigid substrate. Multiple nanotubes can be linked to form the networks and circuits.
Trau et al., "Field-Induced Layering of Colloidal Crystals," Science 272: 706709 (1996) discloses an electrohydrodynamic method for preparing a precise assembly of two- and three-dimensional colloidal crystals on electrode surfaces. The technique disclosed uses electrophoresis, with deposition and arrangement of the particles on the electrode. The technique provides for mono- or multi-layer crystalline films. It is also mentioned that the technique may be used to assemble macromolecules such as proteins into two dimensional crystals.
Monnier et al., "Cooperative Formation of Inorganic-Organic Interfaces in the Synthesis of Silicate Mesostructures," Science 261: 1299-1303 (1993)) discusses a theoretical model of the formation and morphologies of surfactant silicate mesostructures. The article proposes a model for the transformation of a surfactant silicate system from the lamellar mesophase to the hexagonal mesophase. The effect of pH and ionic strength on mesophase structure are also discussed.
In a recent development, U.S. Pat. Nos. 5,304,382, 5,358,722, and 5,491,219, disclose the use of apoferritin devoid of ferrihydride as another solution to the problem of producing small particles. These ferritin analogs consist of an apoferritin shell and an inorganic core, and are thought to be useful in the production of ultrafine particles for high performance ceramics, drug delivery, and other uses.
Ferritin is a protein involved in the regulation of iron in biological systems. In nature, ferritin consists of a protein shell, having 24 structurally equivalent protein subunits surrounding a near spherical core of hydrous ferric oxide ("ferrihydrite"). The core is disclosed as being any organic or inorganic material with the exception of ferrihydrite. Once a core has formed in the process of these patents, the protein coat can be removed and the freed core particles isolated. The process is disclosed as providing for particles approximately 5 to 8 nanometers in diameter. However, this system is size constrained, such that homogeneous particles of smaller or larger sizes are not possible.
A general review of systems employing the apoferritin/core nanoscale particle production system is provided by Douglas, "Biomimetic Synthesis of Nanoscale Particles in Organized Protein Cages," Biomimetic Materials Chemistry, S. Mann (ed.) VCH Publishers, New York (1996).
Additional information on the apoferritin/core system includes:
Douglas et al., "Inorganic-Protein Interactions in the Synthesis of a Ferromagnetic Nanocomposite," American Chemical Society, ACS Symposium Series: Hybrid Organic-Inorganic Composites, J. Mark, C. Y-C Lee, P. A. Bianconi (eds.) (1995) discloses the preparation of a ferrimagnetic iron oxide-protein composite comprising an apoferritin shell and iron oxide core. The core is said to consist of magnetite or maghemite, but was thought to be predominantly maghemite. This magnetoferritin is said to be ideal for bio-compatible nmr imaging, and other biological and medical applications.
Douglas et al., "Synthesis and Structure of an Iron(III) Sulfide-Ferritin Bioinorganic Nanocomposite," Science 269: 54-57 (1995) discloses production of iron sulfide cores inside ferritin shells via an in situ synthesis reaction. The cores are disclosed as a mostly amorphous sulfide consisting predominantly of Fe(III). Cores are described as a disordered array of edge-shared FeS.sub.2 units. Native ferritin particles with sulfided cores are taught to contain between 500 and 3000 iron atom cores, most predominantly in the Fe(III) form. Douglas et al. further disclose that the biomimetic approach to the production of nanoparticles may be useful for biological sensors, drug carriers, and diagnostic and bioactive agents.
Bulte et al., "Magnetoferritin: Characterization of a Novel Superparamagnetic MR Contrast Agent," JMRI, May/June 1994, pp. 497-505, discloses use of horse spleen apoferritin to prepare nanoparticles having a ferritin shell and iron oxide core. The article discloses that novel materials with defined crystal size can be produced by "confined biomineralization within specific subunit compartments." The magnetoferritin produced in the technique described is said to be useful in the production of a nanometer-scale contrast agent for magnetic resonance imaging. Coupling of "bioactive substances" to the ferritin case is further disclosed. Such substances are taught to include antibody fragments and synthetic peptides, which may be useful in tissue-specific imaging.
Meldrum et al., "Magnetoferritin: In Vitro Synthesis of a Novel Magnetic Protein," Science 257: 522-523 (1992) discloses the preparation of magnetoferritin by incubation of apoferritin in a solution of Fe(II) and with slow oxidation. The process described resulted in the discrete, spherical nanometer (ca. 6.0 nm) core particles surrounded by a ferritin protein shell. The core was consistent with being either magnetite or maghemite, most likely magnetite. Possible uses for the magnetoferritin particles are disclosed as the following: (1) industrial applications, (2) study of magnetic behavior as a function of miniaturization, (3) elucidation of iron oxide biomineralization processes, (4) magnetic imaging of biological tissue, and (5) in separation procedures involving cell and antibody labeling.
Meldrum et al., "Reconstitution of Manganese Oxide Cores in Horse Spleen and Recombinant Ferritin," Journal of Inorganic Biochemistry, 58: 59-68 (1995) discloses the formation of MnOOH cores within the nanoscale cavity of ferritin. Ferritin reconstitution with MnOOH cores is taught to be a nonspecific pathway, and an "all or nothing effect" (i.e., either unmineralized or fully loaded). Different apoferritin sources were used: (1) horse spleen ferritin, (2) recombinant H- and L-chain homopolymers and (3) H-chain variants containing site-directed modifications at the ferroxidase and putative Fe nucleation centers. The particle cores are described as being amorphous, whereas particles formed in bulk solution under substantially the same conditions were crystalline.
Bulte et al., "Initial Assessment of Magnetoferritin Biokinetics and Proton Relations Enhancement in Rats," Acad. Radiol., 2: 871-878 (1995), discloses blood clearance, in vivo biodistribution and proton relaxation enhancement of magnetoferritin (1.4 mg Fe/kg) in nude rats carrying a xenografted human small cell lung carcinoma. The kinetics of blood clearance was biexponential with an initial half-life of 1.4 to 1.7 min and a longer component lasting several hours. Ex vivo relaxometry revealed uptake in the liver, spleen and lymph nodes when magnetoferritin was administered with or without a pre-injection of apoferritin. No involvement with ferritin receptors (displayed on the carcinoma) was seen. Magnetoferritin is said to be potentially useful as an imaging agent for liver, spleen and lymph nodes.
Bulte et al., "Magnetoferritin: Biomineralization as a Novel Molecular Approach in the Design of Iron-Oxide-Based Magnetic Resonance Contrast Agents," Investigative Radiobiology 20 (Supplement 2): S214-S216 (1994) reports on the magnetometry and magnetic resonance relaxometry of magnetoferritin. Magnetoferritin is described as a biocompatible magnetic resonance contrast agent. The publication further discloses that magnetoferritin has a convenient matrix for complexing a wide variety of bioactive substances and may provide a basis for a novel generation of biocompatible magnetopharmaceuticals.
However, as mentioned above, in each of these systems employing the apoferritin/core system, whereas the distribution of the nanoscale particles is substantially homogeneous, the size of the particles is constrained by the size of the ferritin cavity. Furthermore, the apoferritin/core system is restricted such that large molecules may not readily enter the protein. Additionally, the internal cavity of ferritin is restricted in size 8 nm or less.
In light of the state of the art, then, it is one object of the invention to provide new and improved nanoscale organic, inorganic and/or organo-metallic particles useful in high performance materials, such as ceramics, lubricants, semiconductors and catalysts.
Another object of the invention is to provide new and improved nanoscale organic, inorganic and/or organo-metallic particles useful in drug delivery, medical imaging and other medical applications.
Another object of the invention is to provide for new and improved nanoscale particles having a substantially homogeneous size distribution and a predetermined, preselected particle size.
Another object of the invention is to provide for methods of producing nanoscale particles through controlled gating of a virion capsid, allowing the controlled entrapment and/or release of organic, inorganic and/or organo-metallic nanoparticles.
Other objects of the invention include the methods of making organic, inorganic and organo-metallic nanoscale particles having a defined and homogeneous size distribution and shape, as well as methods of using the nanoscale particles so produced in the manufacture of useful articles and devices. These and other objects of the invention will become readily apparent to those skilled in the art from the detailed description of the invention that follows.